Nearly all animal cells maintain a large difference in sodium concentrations between their interiors (typically 10-40 mM) and the extracellular milieu (120-450 mM). This gradient is used to power nutrient uptake, epithelial transport, regulation of other intracellular ions, and transmission of electrical impulses. These functions are so important that organisms devote a major part of their metabolic energy to maintaining the sodium gradient (1,2).
Measurements of intracellular Na.sup.+ are essential to understanding the many biological roles of this ion. Current techniques fall into three categories: (1) Assays that measure total cell Na.sup.+ but destroy the tissue; (2) Non-destructive assays that rely on nuclear magnetic resonance; and (3) Non-destructive assays that rely on well-defined physiochemical equilibria to measure free Na.sup.+ ! or Na.sup.+ activity.
Examples of assays that measure total cell Na.sup.+ but destroy the tissue include flame photometry, atomic absorption, neutron activation, counting of .sup.22 Na at isotopic equilibrium, and electron microprobe analysis (30). The destructive nature of these techniques is obviously a drawback when time courses are desired. Except for the electron-microscopic methods, these techniques lack spatial resolution and demand careful removal of extracellular fluid, which usually has a much higher concentration of Na.sup.+ than the cells. The most general problem (18,31,32) is that the total intracellular Na.sup.+ ! usually considerably exceeds free Na.sup.+ !.sub.i, and it is the latter that affects binding equilibria, transmembrane electrochemical gradients, and cell function. Free and total Na.sup.+ ! are known to be able to vary independently (32).
It is well known that NMR techniques using dysprosium shift reagents can quantify the amount of intracellular Na.sup.+ that is readily exchangeable on the NMR time scale (33,34). This probably includes weakly bound Na.sup.+ as well as free. Though non-destructive, this technique requires relatively large amounts of tissue packed at high density in a magnet cavity, an environment awkward for other manipulations.
Techniques that rely on well-defined physiochemical equilibria to nondestructively measure free Na.sup.+ ! (or Na.sup.+ activity) include .sup.19 F NMR of Na.sup.+ -sensitive chelators (6), Na.sup.+ -selective microelectrodes (32), and the new fluorescent indicators of the present invention. Advantages of fluorescent indicators include excellent spatial and unsurpassed temporal resolution, compatibility with cell types too small or fragile to impale with ion-selective and voltage reference barrels, and applicability to single cells as well as to populations (35). Perhaps the chief disadvantage is the demand for optical clarity of the tissue.
Prior art techniques for measuring and manipulating intracellular free sodium concentrations (Na.sup.+ !.sub.i) have been severely limited by the lack of synthetic ligands that can bind sodium with the requisite affinity and specificity in aqueous solution at pH 7. A particularly desirable ligand would have the following properties:
(1) Na.sup.+ should bind with a dissociation constant (K.sub.d) of 5-50 mM at pH 7, in aqueous solution with no organic co-solvents permitted. Such a K.sub.d would approximately match the expected range for Na.sup.+ !.sub.i and maximize sensitivity to small changes in Na.sup.+ !.sub.i. Excessive Na.sup.+ affinity would be undesirable, since the indicator would then either be Na.sup.+ -saturated and unresponsive, or if applied in excess would depress Na.sup.+ !.sub.i. PA1 (2) The indicator should have enough discrimination against K.sup.+ (at least twenty-fold, or a K.sub.d &gt;150 mM), H.sup.+ (highest pK.sub.a &lt;6.5), Mg.sup.2+ (K.sub.d &gt;10 mM), and Ca.sup.2+ (K.sub.d &gt;10 .mu.M) so that physiological variations in those ions have little effect. PA1 (3) It would show reasonably strong fluorescence, characterizable by a product of extinction coefficient and fluorescence quantum yield exceeding 10.sup.3 M.sub.-1 cm.sub.-1. PA1 (4) Its excitation wavelengths should exceed 340 nm, because shorter wavelengths demand expensive quartz rather than glass microscope optics and are strongly absorbed by nucleic acids and aromatic amino acids. PA1 (5) Emission wavelengths should exceed 500 nm to reduce overlap with tissue autofluorescence from reduced pyridine nucleotides peaking near 460 nm. PA1 (6) Either the excitation or emission spectrum or both should undergo a large wavelength shift upon binding Na.sup.+, so that ratioing of signals at two excitation or two emission wavelengths can cancel out the local pathlength, dye concentration, and wavelength-independent variations in illumination intensity and detection efficiency. PA1 (7) The indicator should have enough polar groups such as carboxylates to render it highly water-soluble and impermeant through membranes, so that it does not rapidly leak out of cells. PA1 (8) The polar groups just mentioned should be maskable by nonpolar protecting groups hydrolyzable by cytoplasm, so that large populations of cells can be loaded with the indicator by incubating them with the membrane-permanent nonpolar derivative rather than requiring microinjection or other techniques of membrane disruption. The most obvious protecting groups are acetoxymethyl esters, which have proven to be useful with a wide variety of cation indicators (51,52).
No such compound has yet been demonstrated to work in living cells despite nearly two decades worth of research on crown ethers and related ligands. Indicator dyes with visible absorbance and moderate preference for Na.sup.+ over K.sup.+ have been reported (3,4) but their operation is limited to nonaqueous solvents like acetonitrile, and no quantitative data is available on their cation binding constants. Higher affinity and selectivity for Na.sup.+ over K.sup.+ in water can be obtained with macrobicyclic chelators, for example the cryptand "2.2.1!" (see ref. 5 and FIG. 1). Recently, fluorine-substituted cryptands have been introduced for measurement of Na.sup.+ !.sub.i by .sup.19 F-NMR (6). A promising fluorescent version was also described by Smith, et al., (1988) (see ref. 7), but its excitation and emission spectra peaked at rather short wavelengths, 320 and 395 nm respectively, and no demonstration of intracellular use was given. The highest selectivities for Na.sup.+ over K.sup.+ are obtained in very large rigid chelators called "spherands"; so far these require organic solvents for solubility and are so rigid that hours to days are required for equilibration with Na.sup.+ (8,9). The main mechanism by which they give optical shifts upon metal binding has been the displacement of a proton from the binding cavity, but this equilibrium must inherently be pH-sensitive, which is an unwanted feature. We chose to explore crown ethers rather than the more elaborate cryptands and spherands both for ease of synthesis and because of a concern that the conformational rigidity and preorganization of cryptands and spherands would tend to reduce the spectroscopic shift upon metal binding.
The present invention discloses a new series of macrocyclic compounds that can chelate alkali metal cations and that attain the basic goals described above for a desirable fluorescent sodium indicator. Tests in lymphocytes, hepatocytes, fibroblasts (10), smooth muscle cells (11), and gastric glands (12) demonstrate the biological utility of the macrocyclic compounds of the present invention for nondestructive observation of Na.sup.+ !.sub.i in individual cells viewed by fluorescence microscopy.